Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: a potential candidate molecule for immunotherapy in cervical cancer.

BACKGROUND: Dendritic cell (DC)-based immunotherapy is capable of activating the immune system and in particular tumor-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumor. However, major limitations are the availability of autologous tumor cells as antigenic source and the selection of antigen that may have potential to activate both CD4+ and CD8+ T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4+ and CD8+ T cell responses for future DCs-based vaccine trials in cervical cancer patients. METHODS: Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. The efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumor lysates (TLDCs), to induce CD4+, CD8+ T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. RESULTS: Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83; P = 0.4; migration; P = 0.2). In contrast, although TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, this difference was not statistically significant (IL12p40, P = 0.06). Further we also observed that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+ Tregs in autologous co-cultures. CONCLUSIONS: In summary, in order to overcome the limitation of the availability of autologous tumor cells as antigenic sources, our present strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer trials and warrants further studies. This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumor burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention.

Knockdown of A-kinase anchor protein 4 inhibits proliferation of triple-negative breast cancer cells in vitro and in vivo.

Triple-negative breast cancers are the most aggressive subtypes with poor prognosis due to lack of targeted cancer therapy. Recently, we reported an association of A-kinase anchor protein 4 expression with various clinico-pathological parameters of breast cancer patients. In this context, we examined the effect of knockdown of A-kinase anchor protein 4 on cell cycle, apoptosis, cellular proliferation, colony formation, migration, and invasion in triple-negative breast cancer cells. We also examined the synergistic cytotoxic effect of paclitaxel on A-kinase anchor protein 4 downregulated triple-negative breast cancer cells. Knockdown of A-kinase anchor protein 4 resulted in significant reduction in cellular growth and migratory abilities. Interestingly, we also observed enhanced cell death in A-kinase anchor protein 4 downregulated cells treated with paclitaxel. Knockdown of A-kinase anchor protein 4 in cell cycle resulted in G0/G1 phase arrest. Knockdown of A-kinase anchor protein 4 also led to increased reactive oxygen species generation as a result of upregulation of NOXA and CHOP. In addition, levels of cyclins, cyclin-dependent kinases, anti-apoptotic molecules, and mesenchymal markers were reduced in A-kinase anchor protein 4 downregulated cells. Moreover, downregulation of A-kinase anchor protein 4 also caused tumor growth reduction in in vivo studies. These data together suggest that A-kinase anchor protein 4 downregulation inhibits various malignant properties and enhances the cytotoxic effect of paclitaxel, and this combinatorial approach could be useful for triple-negative breast cancer treatment.

Sperm associated antigen 9 (SPAG9) a promising therapeutic target of ovarian carcinoma.

SPAG9 is a novel tumor associated antigen, expressed in variety of malignancies. However, its role in ovarian cancer remains unexplored. SPAG9 expression was validated in ovarian cancer cells by real time PCR and Western blot. SPAG9 involvement in cell cycle, DNA damage, apoptosis, paclitaxel sensitivity and epithelial- mesenchymal transition (EMT) was investigated employing RNA interference approach. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro. Quantitative PCR and Western blot analysis revealed SPAG9 expression in A10, SKOV-3 and Caov3 compared to normal ovarian epithelial cells. SPAG9 ablation resulted in reduced cellular proliferation, colony forming ability and enhanced cytotoxicity of chemotherapeutic agent paclitaxel. Effect of ablation of SPAG9 on cell cycle revealed S phase arrest and showed decreased expression of CDK1, CDK2, CDK4, CDK6, cyclin B1, cyclin D1, cyclin E and increased expression of tumor suppressor p21. Ablation of SPAG9 also resulted in increased apoptosis with increased expression of various pro- apoptotic molecules including BAD, BID, PUMA, caspase 3, caspase 7, caspase 8 and cytochrome C. Decreased expression of mesenchymal markers and increased expression of epithelial markers was found in SPAG9 ablated cells. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro assays which showed that ablation of SPAG9 resulted in increased paclitaxel sensitivity and caused enhanced cell death. In vivo ovarian cancer xenograft studies showed that ablation of SPAG9 resulted in significant reduction in tumor growth. Present study revealed therapeutic potential of SPAG9 in ovarian cancer.

Sperm-associated antigen 9 (SPAG9) promotes the survival and tumor growth of triple-negative breast cancer cells.

Recently, we demonstrated the association of sperm-associated antigen 9 (SPAG9) expression with breast cancer. Among breast cancer, 15 % of the cancers are diagnosed as triple-negative breast cancers (TNBC) based on hormone receptor status and represent an important clinical challenge because of lack of effective available targeted therapy. Therefore, in the present investigation, plasmid-based small hairpin (small hairpin RNA (shRNA)) approach was used to ablate SPAG9 in aggressive breast cancer cell line model (MDA-MB-231) in order to understand the role of SPAG9 at molecular level in apoptosis, cell cycle, and epithelial-to-mesenchymal transition (EMT) signaling. Our data in MDA-MB-231 cells showed that ablation of SPAG9 resulted in membrane blebbing, increased mitochondrial membrane potential, DNA fragmentation, phosphatidyl serine surface expression, and caspase activation. SPAG9 depletion also resulted in cell cycle arrest in G0-G1 phase and induced cellular senescence. In addition, in in vitro and in vivo xenograft studies, ablation of SPAG9 resulted in upregulation of p21 along with pro-apoptotic molecules such as BAK, BAX, BIM, BID, NOXA, AIF, Cyto-C, PARP1, APAF1, Caspase 3, and Caspase 9 and epithelial marker, E-cadherin. Also, SPAG9-depleted cells showed downregulation of cyclin B1, cyclin D1, cyclin E, CDK1, CDK4, CDK6, BCL2, Bcl-xL, XIAP, cIAP2, MCL1, GRP78, SLUG, SNAIL, TWIST, vimentin, N-cadherin, MMP2, MMP3, MMP9, SMA, and ß-catenin. Collectively, our data suggests that SPAG9 promotes tumor growth by inhibiting apoptosis, altering cell cycle, and enhancing EMT signaling in in vitro cells and in vivo mouse model. Hence, SPAG9 may be a potential novel target for therapeutic use in TNBC treatment.

Heat shock protein 70-2 (HSP70-2) is a novel therapeutic target for colorectal cancer and is associated with tumor growth.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer related deaths worldwide both in men and women. Our recent studies have indicated an association of heat shock protein 70-2 (HSP70-2) with bladder urothelial carcinoma. In the present study, we investigated the association of HSP70-2 with various malignant properties of colorectal cancer cells and clinic-pathological features of CRC in clinical specimens. METHODS: HSP70-2 mRNA and protein was investigated expression by RT-PCR, immunohistochemistry, immunofluorescence, flow cytometry and Western blotting in CRC clinical specimens and COLO205 and HCT116 cell lines. Plasmid-based gene silencing approach was employed to study the association of HSP70-2 with various malignant properties of COLO205 and HCT116 cells in in vitro and with tumor progression in in vivo COLO205 human xenograft mice model. RESULTS: HSP70-2 expression was detected in 78 % of CRC patients irrespective of various stages and grades by RT-PCR and IHC. Our analysis further revealed that HSP70-2 expression was detected in both COLO205 and HCT116 cell lines. Ablation of HSP70-2 expression resulted in reduced cellular growth, colony forming ability, migratory and invasive ability of CRC cells. In addition, ablation of HSP70-2 expression showed significant reduction in tumor growth in COLO205 human xenograft in in vivo mouse model. CONCLUSION: Collectively, our results indicate that HSP70-2 is associated with CRC clinical specimens. In addition, down regulation of HSP70-2 expression reduces cellular proliferation and tumor growth indicating that HSP70-2 may be a potential therapeutic target for CRC treatment.

Targeting the testis-specific heat-shock protein 70-2 (HSP70-2) reduces cellular growth, migration, and invasion in renal cell carcinoma cells.

Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiotherapy and chemotherapy. Current therapies for the RCC patients are limited owing to lack of diagnosis and therapeutic treatments. Testis-specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, has been shown to be associated with various cancers. In the present study, we investigated the putative role of HSP70-2 in various malignant properties of the RCC cells. HSP70-2 messenger RNA (mRNA) and protein expression was investigated in A704, ACHN, and Caki-1 cells derived from the RCC patients. We assessed the expression of HSP70-2 gene and protein by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The expression of HSP70-2 protein was further validated by performing indirect immunofluorescence (IIF) and flow cytometry. The malignant properties of high-grade invasive A704 and Caki-1 cells, such as cellular proliferation, colony formation, migration, invasion, and wound healing, were evaluated by silencing the expression of HSP70-2 gene in these cells. Statistical significance was defined using Student's t test. Our RT-PCR and Western blotting data showed the expression of HSP70-2 in all RCC cells. Our results showed that HSP70-2 was predominantly expressed in cytoplasm and found to be colocalized with endoplasmic reticulum, mitochondria, Golgi body, and plasma membrane but not the nuclear envelope. Knockdown of HSP70-2 expression with specific short hairpin RNA (shRNA) demonstrated significant reduction in cell growth and colony formation. Further, a marked reduction in cell migration and invasion was also observed, indicating the potential role of HSP70-2 in metastasis. Collectively, our data suggest that HSP70-2 plays a key role in cancerous growth and invasive potential of RCC cells. Thus, HSP70-2 could serve as a novel potential therapeutic target for the RCC.

Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

Expression and humoral response of A-kinase anchor protein 4 in cervical cancer.

BACKGROUND: Cervical cancer is one of the major gynecologic cancers. In developing countries, because of a lack of medical support and infrastructure, cervical cancer is the leading cause of cancer-related deaths. Therefore, there is a need to identify novel biomarkers for cervical cancers. In this context, cancer-testis (CT) antigens represent a unique class of tumor antigens that have been shown to be associated with various solid tumors. These antigens have restricted expression in testis and no expression in somatic tissues. Because of their restricted expression, CT antigens are novel candidate molecules for early detection and diagnosis and immunotherapy. In the present study, novel CT antigen A-kinase anchor protein 4 (AKAP4) expression and humoral response were investigated in patients with cervical cancer. METHODS: In this study, 74 cervical cancer tissue specimens, which included different tumor stages (stage I [n = 35], stage II [n = 39]) and histologic grades (grade 1 [n = 17], grade 2 [n = 46], and grade 3[n = 11]) and 62 adjacent noncancerous tissue specimens were investigated for AKAP4 gene expression by using reverse transcriptase polymerase chain reaction and in situ RNA hybridization. Furthermore, AKAP4 protein expression was determined by immunohistochemistry. In addition, humoral response against purified recombinant AKAP4 protein was determined in available sera of 70 patients with cervical cancer by enzyme-linked immuno assay (ELISA). FINDINGS: Our study revealed that AKAP4 gene and protein expression was detected in 86% of total patients with cervical cancer. Based on the AKAP4 immunoreactivity score, most of stage I (n = 22/29) and stage II (n = 30/35) specimens revealed high AKAP4 expression (≥50% AKAP4-positive cells). A-kinase anchor protein 4 expression was significantly associated with early grades tumor specimens (P = 0.023). In addition, humoral response was detected in 53% of patients irrespective of stages, lymph node positivity, and grades. CONCLUSIONS: Collectively, our data indicate the putative role of AKAP4 in early tumorigenesis and may be implicated as a biomarker and immunotherapeutic target for cervical cancer.

Sperm-associated antigen 9 is a novel biomarker for colorectal cancer and is involved in tumor growth and tumorigenicity.

Colorectal cancer (CRC) is the second most common tumor in developed countries. The present study was undertaken to determine the expression of the sperm-associated antigen 9 gene (SPAG9) as a possible biomarker in CRC, to investigate its correlation with humoral immune response and different stages and grades in CRC patients, and to explore its possible role in colon tumorigenesis in vitro and in an in vivo mouse model. SPAG9 expression was determined by RT-PCR, in situ RNA hybridization, and immunohistochemistry. Humoral response against SPAG9 was detected by enzyme-linked immunosorbent assay and Western blotting. SPAG9 gene silencing was performed using plasmid-based small interfering RNA to study various malignant properties of colon cancer cells in vitro and in vivo. The majority of CRC patients showed SPAG9 expression and generated humoral response. There was a close relationship between SPAG9 protein expression and humoral immune response in the majority of early-stage CRC patients, indicating that anti-SPAG9 antibodies could be a novel serum biomarker for early diagnosis. The down-regulation of SPAG9 (mediated by small interfering RNA) inhibited malignant properties in in vitro and significantly suppressed tumor growth in vivo. These findings collectively suggest that SPAG9 may have a role in tumor development and early spread and thus could serve as a novel target for early detection and for cancer immunotherapy.

Formylation of single-walled carbon nanotubes.

We report an improved, elegant method for the covalent formylation of single-wall carbon nanotubes (SWNTs) via formyl transfer from N-formylpiperidine, which could potentially open the gateway for more versatile chemical modification of carbon nanotube (CNT) walls than is possible via other reported functionalisation methods. The formylation reaction does not inflict damage upon the pristine CNT structure, unlike the currently commonly used carboxylation route, and involves much fewer steps, and takes considerably less time, than most other reported routes. The modified SWNTs have been characterised by Raman spectroscopy, ultraviolet-visible-near infrared (UV-vis-NIR) spectroscopy and "covalent tagging" with derivatising groups followed by thermogravimetric analysis-mass spectroscopy (TGA-MS). UV-vis-NIR spectroscopy shows that there is only limited disruption of the intrinsic electronic structure of the SWNTs. This is confirmed from estimates of the extent of functionalisation from TGA-MS, which suggest that it may be as low as 2 atomic per cent.

Effect of Temperature and Branched Crosslinkers on Supported Graphene Oxide Pervaporation Membranes for Ethanol Dehydration.

We describe the performance of graphene oxide (GO) membranes stabilized by crosslinkers and supported on polyethersulfone films in the dehydration of ethanol in a continuous cross-flow pervaporation set-up. We used two crosslinker species with branched structures (humic acid-like substances derived from urban waste and a synthetic hyperbranched polyol). The supported crosslinked GO films were prepared by rod coating on a polyethersulfone ultrafiltration membrane. Pervaporation experiments were carried out at temperatures of 40, 50, 60 and 70 °C. When the feed comprised pure water and ethanol, a much higher flux of water than ethanol was observed at all temperatures through GO films stabilized by the two crosslinkers (humic acid, GO-HAL, and the synthetic hyperbranched polyol, GO-HBPO), indicating the separation ability of these crosslinked membranes. For feed mixtures of water and ethanol, the GO-HAL and GO-HBPO membranes showed good separation performances by producing permeates with a significantly higher water content than the feed at all temperatures.

Erratum: Heat shock protein 70-2 (HSP70-2) a novel cancer testis antigen that promotes growth of ovarian cancer.

[This corrects the article on p. 1252 in vol. 7, PMID: 28670489.].

Sperm-associated antigen 9, a novel biomarker for early detection of breast cancer.

To date, there have been no tumor biomarkers validated and incorporated into oncologic practice for the early diagnosis of breast cancer. Recently, we showed that sperm-associated antigen 9 (SPAG9), a member of cancer testis (CT) antigen family, is associated with ovarian carcinomas. In the present study, we investigated SPAG9 expression and humoral immune response in breast cancer. We further evaluated the diagnostic potential of autoantibodies to SPAG9 protein in various stages, grades, and histotypes of breast cancer. We analyzed the association of SPAG9 immunoreactivity score (IRS) with predicted risk of breast cancer recurrence over 10 years. Our reverse transcription-PCR and immunohistochemical analyses revealed SPAG9 expression in 88% breast cancer specimens independent of tumor stages and grades. Further, the humoral immune response against SPAG9 was detected in 80% breast cancer patients with SPAG9-expressing tumors. The linear regression modeling predicted a direct relationship between presence of lymphovascular invasion and high SPAG9 IRS, whereas the univariate and multivariate logistic regression models predicted a strong association of SPAG9 IRS with tumor grade. Further, our data indicated a significant higher trend of SPAG9 IRS with the predicted high risk of breast cancer recurrence. The present investigation reports for the first time SPAG9 expression and humoral immune response in early stages and low-grade breast cancer. Although our data indicated that autoantibodies against SPAG9 represent a promising approach for the development of biomarker, further large-scale validation studies are required to establish its potential use in early diagnosis and monitoring of breast cancer recurrence.

Sperm-associated antigen 9, a novel cancer testis antigen, is a potential target for immunotherapy in epithelial ovarian cancer.

PURPOSE: Cancer testis antigens are a group of tumor antigens with gene expression restricted to male germ cells in the testis and in various cancerous tissues. Recently, we reported a novel testis-specific sperm-associated antigen 9 (SPAG9) gene, a new member of the c-Jun NH(2)-terminal kinase-interacting protein family, having functional role in sperm-egg fusion and mitogen-activated protein kinase signaling pathway. National Center for Biotechnology Information Blast searches revealed SPAG9 nucleotide sequence similarities with expressed sequence tags of various cancerous tissues. In an effort to examine the clinical utility of SPAG9, we investigated the SPAG9 mRNA and protein expression in epithelial ovarian cancer (EOC). Humoral immune response to SPAG9 was also evaluated in EOC patients. EXPERIMENTAL DESIGN: We determined the expression profile of SPAG9 transcript by reverse transcription-PCR and RNA in situ hybridization and SPAG9 protein expression by immunohistochemistry in EOC specimens and human ovarian cancer cell lines. Using ELISA and Western blotting, we analyzed specific antibodies for SPAG9 in sera from patients with EOC. RESULTS: SPAG9 mRNA and protein expression was detected in 90% of EOC tissues and in all three human ovarian cancer cell lines. Specific SPAG9 antibodies were detected in 67% of EOC patients and not in sera from healthy individuals. CONCLUSIONS: Our findings indicate that SPAG9 is highly expressed in EOC and immunogenic in patients. Humoral immune response against SPAG9 in early stages of EOC suggests its important role in early diagnostics. These results collectively suggest that SPAG9, a novel member of cancer testis antigen family, could be a potential target for the development of diagnostic and therapeutic methods in EOC.

Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv.

Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of D-glucopyranose-6-phosphate to D-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 A and belonged to the orthorhombic space group I2(1)2(1)2(1), with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 A.

Role of A-Kinase anchor protein (AKAP4) in growth and survival of ovarian cancer cells.

Ovarian cancer represents one of the most common malignancies among women with very high mortality rate worldwide. A-kinase anchor protein 4 (AKAP4), a unique cancer testis (CT) antigen has been shown to be associated with various malignant properties of cancer cells. However, its involvement in various molecular pathways in ovarian cancer remains unknown. In present investigation, employing gene silencing approach, we examined the role of AKAP4 in cell cycle, apoptosis and epithelial-mesenchymal transition (EMT). Further, we also investigated the effect of ablation of AKAP4 on tumor growth in SCID mice ovarian cancer xenograft mouse model. Our results showed that ablation of AKAP4 resulted in increased reactive oxygen species (ROS) generation, DNA damage, cell cycle arrest and apoptosis in ovarian cancer cells. AKAP4 knockdown lead to degradation of protien kinase A (PKA) which was rescued by proteosome inhibitor MG-132. ROS quencher N-acetyl cysteine (NAC) treatment rescued cell cycle arrest and resumed cell division. Subsequently, increased expression of pro-apoptotic molecules and decreased expression of pro-survival/anti-apoptotic factors was observed. As a result of AKAP4 depletion, DNA damage response proteins p-γH2AX, p-ATM and p21 were upregulated. Also, knockdown of CREB resulted in similar findings. Further, PKA inhibitor (H89) and oxidative stress resulted in similar phenotype of ovarian cancer cells as observed in AKAP4 ablated cells. Collectively, for the first time our data showed the involvement of AKAP4 in PKA degradation and perturbed signaling through PKA-CREB axis in AKAP4 ablated ovarian cancer cells.

Heat shock protein 70-2 (HSP70-2) a novel cancer testis antigen that promotes growth of ovarian cancer.

Heat shock protein 70-2 (HSP70-2) is known to be involved in tumor progression. However, its molecular role and mechanism in epithelial ovarian cancer (EOC) remains unknown. In the present investigation, we examined the role of HSP70-2 in cell cycle, apoptosis and epithelial mesenchymal transition pathways in EOC cells in in vitro and in-vivo xenograft mouse model. To investigate the role of HSP70-2 in ovarian cancer, plasmid driven short hairpin RNA approach was used to examine HSP70-2 gene and protein expression in ovarian cancer cell line A-10 (origin: serous papillary cystadenocarcinoma), Caov-3 (origin: adenocarcinoma) and SKOV3 (origin: adenocarcinoma; derived from metastatic site: ascites) by RT-PCR, quantitative-PCR, immunohistochemistry and Western blotting. Light microscopy, scanning electron microscopy, viability tests, and flow cytometry were used to study the cellular proliferation, onset of senescence, colony forming ability and morphological features of cancer cells. Cell migration and invasion ability was evaluated by wound healing and Boyden chamber assays. Further, we studied the effect of HSP70-2 protein ablation on human ovarian xenograft mice model. At molecular level, various molecules involved in apoptosis, cell cycle and epithelial-mesenchymal-transition were also examined both in in-vitro and in-vivo xenograft mouse model. The knockdown of HSP70-2 expression by gene silencing resulted in the onset of apoptosis, senescence, reduced cellular growth and colony forming ability of EOC cells. Interestingly, the migration, invasion and wound healing abilities of cells were also significantly inhibited. In addition, the ablation of HSP70-2 resulted in the upregulation of cytochrome-C, caspase 3, caspase 7, caspase 9, APAF1, BAX, BIM, BAK, BAD, BID, PUMA, NOXA, p16, p21, Rb, E-cadherin, cytokeratin 18, EMA in these cells as well as in the xenograft tumor specimens. However, there was downregulation of PARP1, BCL-2, Bcl-xL, MCL-1, Survivin, XIAP, cIAP2, CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin E, cyclin A2, cyclin B1, p-Rb, N-cadherin, SNAIL, SLUG, VIMENTIN, SMA, MMP2, MMP3, MMP9 and TWIST in these samples. Furthermore, the xenograft studies showed significant reduction in the tumor growth. Our results suggest that HSP70-2 can promote cellular growth and invasion of EOC cells and therefore may be a potential therapeutic target in EOC.

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Spindle Cell Variant of Embryonal Rhabdomyosarcoma: A Rare Entity with Diagnostic Challenges.

The spindle cell variant of embryonal rhabdomyosarcoma is a rare and a better differentiated variant of embryonal rhabdomyosarcoma, having a better prognosis compared to other types of rhabdomyosarcomas. So, it needs to be distinguished from classical forms of the neoplasm. Its morphological resemblance to spindle cell neoplasms like leiomyosarcomas and fibrosarcomas may pose diagnostic difficulties for the pathologist. This problem can be overcome by careful search for rhabdomyoblasts in sections, which are usually few, and Immunohistochemistry for myogenin. In the present case, a 15-year-old female presented with a progressively increasing swelling in the right upper eyelid, which was diagnosed as a rare variant of rhabdomyosarcoma. We have also attempted to discuss its differential diagnosis, and to emphasize the fact that this rare entity may be misdiagnosed.